中文摘要：

目的检测裸露角质蛋白同源物2（nakedcuticlehomo／og2，Nkd2）在大鼠牙根形成和牙囊细胞成骨向分化过程中的表达变化，为探讨Nkd2在牙囊细胞成骨向分化中的具体分子机制提供实验基础。方法免疫组化法检测Nkd2在SD大鼠出生后1、3、5、7、9、11及13d下颌第一磨牙牙胚近基底侧牙囊组织中的表达。茜素红染色和十六烷基吡啶观察分析大鼠牙囊细胞（dentalfolliclecellsofrat，rDVC）矿化诱导l、2、3周钙化结节形成情况。蛋白质印迹法分析rDFC矿化诱导1、2、3周Nkd2蛋白表达变化及其与成骨因子碱性磷酸酶（alkalinephosphatase，ALP）、Runt相关转录因子2（Runt．relatedtranscriptionfactor-2，RUNX2）和骨钙蛋白的关系。小干扰RNA（smallinterferingRNA，siRNA）沉默rDFC中Nkd2（siRNA干扰组，Si组），经矿化诱导1周，蛋白质印迹法和实时荧光定量PCR（quantitativereal—timePCR，qPCR）检测Si组、阴性对照组（阴性对照RNA组，negativecontrolRNAgroup，Nc组）和空白对照组（mockcontrolgroup，Mock组）Nkd2及其mRNA与ALP、RUNX2和骨钙蛋白的表达变化。结果免疫组化结果显示，大鼠牙根形成中Nkd2在下颌第一磨牙牙胚基底侧牙囊组织中的表达呈现时间差异。随rDFC成骨诱导时间增加，茜素红染色矿化结节逐渐增多，十六烷基吡啶吸光度值逐渐增高（0、1、2、和3周吸光度值分别为0．017±0．005、0．702±0．044、1．812±0．531及2．767±0．253）。蛋白质印迹法结果显示，矿化诱导早期矿化组Nkd2（1．60±0．23）较对照组（1）表达显著上调（P〈0．05），Nkd2表达趋势与成骨相关因子表达趋势一致。siRNA干扰rDFC矿化诱导1周后si组较Nc、Mock组Nkd2和成骨因子在蛋白和mRNA水平显著降低（P〈O．05），Nc与Mock组Nkd2和成骨因子表达差异无统计学意义（P〉0．05）。Si、Nc和Mock组蛋白相对表达量分别为Nkd2：0．42±0．10、1．12±0．07、1?

英文摘要：

Objective To examine the expression of naked cuticle homolog 2 （Nkd2） in the process of root development and osteogenic differentiation of dental follicle cells of rat （rDFC）, in order to explore the molecular mechanisms of Nkd2 on the osteoblast differentiation of rDFCs. Methods Immunohistochemical analysis was used to detect the expression of Nkd2 in the base dental follicle of the mandibular first molar of rat at 1, 3, 5, 7, 9, 11 and 13 days postnatal. Mineralization nodule formation of rDFCs was detected by alizarin red staining and cetylpyridine. The change of Nkd2 during osteogenic differentiation of rDFCs was evaluated by Western blotting and the associations between Nkd2 and osteogenic cytokines of alkaline phosphatase （ALP）, Runt-related transcription factor-2 （RUNX2） and osteocalein （OCN） were examined. The rDFCs were transfeeted with small interfering RNA （siRNA） to knock down the expression of Nkd2 and Western blotting and quantitative real-time PCR （qPCR） were adopted to explore the effects of Nkd2 on osteogenic differentiation by detecting variations of Nkd2 and osteogenic factors ALP, RUNX2, OCN among silencing group （Si）, negative control RNA group （Nc） and mock control group （Mock）, respectively. Results The expression of Nkd2 in the base dental follicle of the mandibular first molar of rat was time dependent. Mineralization nodules of rDFCs and absorbance of cetylpyridine after osteogenic induction increased gradually （the absorbances of cetylpyridine were 0 week： 0.017± 0.005, 1 week： 0.702±0.044, 2 weeks： 1.812±0.531, 3 weeks： 2.767±0.253, respectively）. Results of Western blotting slaowed that Nkd2 （1.60±0.23） of mineralization group was significantly higher than that of control group （1） （P〈0.05） at the early stage of osteogenic differentiation along with the expression of other osteogenic factors. The protein and mRNA of Nkd2 and osteogenic factors were significantly decreased in Si group compared with Nc and Mock group